The objective of this study is to elucidate the intracellular interactions of the HIV regulatory protein Nef with host cell activities. Early in the HIV-1 infection of CD4 T cells this retrovirus expresses regulatory proteins, with the Nef transcript representing nearly 80% of total viral mRNA. In vivo infectivity by SIV and HIV is achieved in the absence of Nef expression, but there is an absolute requirement for Nef in the production of high viral titers. In the absence of Nef, there is loss of development of the immunological and neurological dysfunctions characteristic of AIDS. The essential activity of the Nef protein in the development of AIDS has not been defined. We have discovered that expression of Nef in human CD4 T cells results in a lowering of the activation threshold. Since activation is essential for viral synthesis, Nef thus has a direct role in increasing viral production. Evidence suggest that the biochemical site affected by Nef is early in the T cell receptor pathway. We had previously established that the Nef protein associates with a member of the p21-activated kinase (PAK) family, which is known to initiate activation pathways as well as alter cytoskeletal structures. Our exploration of early infection of HIV in quiescent CD4 T cells has lead to the discovery that prior to integration, reverse-transcribed HIV DNA generates multiply-spliced transcripts that encode the HIV regulatory proteins. These expressed proteins prime quiescent cells for activation and enhanced viral production. An understanding of the biochemical activity of this virulence factor could lead to new options for treating HIV-infected individuals.